Immunoglobulins (Igs) are composed of two heavy and two light chains, each of which contains an NH2-terminal antigen-binding variable domain and a COOH-terminal constant domain responsible for the effector functions of antibodies. The COOH-terminal domains of Ig heavy chains form the Fc region and are involved in triggering cellular activities through interaction with specific receptors known as Fc receptors (FcRs). Fc receptors for all Ig classes, or isotypes, (e.g., IgG (Fcxcex3R), IgE (Fcxcex5R), IgA (Fcxcex1R), IgM (FcxcexcR) and IgD (Fcxcex4R) have been identified. The different biological activities of antibodies of different isotypes are based in part on their ability to bind to different FcR expressed on different immune (effector) cells (Fridman, W. H. (September 1991) The FASEB Journal Vol. 5. 2684-2690). Murine antibodies, which are directed against FcRs have been made (See e.g. U.S. Pat. No. 4,954,617 entitled Monoclonal Antibodies To Fc Receptors for Immunoglobulin G on Human Mononuclear Phagocytes and International Patent Application Publication No. WO 91/05871 entitled Monoclonal Antibody Specific For IgA Receptor).
Murine monoclonal antibodies can be useful as human therapeutics and can be produced free of contamination by human pathogens such as the hepatitis or human immunodeficiency virus. However, use of murine monoclonal antibodies in some human therapies, have resulted in the development of an immune response to the xe2x80x9cforeignxe2x80x9d murine proteins. This response has been termed a human anti-mouse antibody or HAMA response (Schroff, R. et al. (1985), Cancer Res., 45, 879-885) and is a condition which causes serum sickness in humans and results in rapid clearance of the murine antibodies from an individual""s circulation. The immune response in humans has been shown to be against both the variable and the constant regions of murine immunoglobulins.
Recombinant DNA technology can be used to alter antibodies, for example, by substituting specific immunoglobulin regions from one species with immunoglobulin regions from another species. Neuberger et al. (Patent Cooperation Treaty Patent Application No. PCT/GB85/00392) describes a process whereby the complementary heavy and light chain variable domains of an Ig molecule from one species may be combined with the complementary heavy and light chain Ig constant domains from another species. This process may be used to substitute the murine constant region domains to create a xe2x80x9cchimericxe2x80x9d antibody which may be used for human therapy. A chimeric antibody produced as described by Neuberger et al. has a human Fc region for efficient stimulation of antibody mediated effector functions, such as complement fixation, but still has the potential to elicit an immune response in humans against the murine (xe2x80x9cforeignxe2x80x9d) variable regions.
Winter (British Patent Application Number GB2188538A) describes a process for altering antibodies by substituting the complementarity determining regions (CDRs) with those from another species. This process may be used to substitute the CDRs from the murine variable region domains of a monoclonal antibody with desirable binding properties (for instance to a human pathogen) into human heavy and light chain Ig variable region domains. These altered Ig variable regions may then be combined with human Ig constant regions to create antibodies which are totally human in composition except for the substituted murine CDRs. The xe2x80x9creshapedxe2x80x9d or xe2x80x9chumanizedxe2x80x9d antibodies described by Winter elicit a considerably reduced immune response in humans compared to chimeric antibodies because of the considerably less murine components. Further, the half life of the altered antibodies in circulation should approach that of natural human antibodies. However, as stated by Winter, merely replacing the CDRs with complementary CDRs from another antibody which is specific for an antigen such as a viral or bacterial protein, does not always result in an altered antibody which retains the desired binding capacity. In practice, some amino acids in the framework of the antibody variable region interact with the amino acid residues that make up the CDRs so that amino acid substitutions into the human Ig variable regions are likely to be required to restore antigen binding.
Bispecific molecules, (e.g., heteroantibodies) comprising an anti-Fc receptor portion and an anti-target portion have been formulated and used therapeutically, e.g., for treating cancer (e.g. breast or ovarian) or pathogenic infections (e.g., HIV) (See, e.g., International Patent Application Publication No. WO 91/05871 entitled Bispecific Heteroantibodies With Dual Effector Functions; and International Patent Application Publication No. WO 91/00360 entitled Bispecific Reagents for AIDS Therapy). In addition, bispecific molecules, which recognize antigens and antigen presenting cells can be administered to a subject to stimulate an immune response (See, e.g., International Patent Application Publication No. WO 92/05793 entitled Targeted Immunostimulation With Bispecific Reagents).
The present invention provides recombinant and chemically synthesized multispecific molecules which target immune cells. The molecules are xe2x80x9cmultispecificxe2x80x9d because they bind to multiple (two or more), distinct targets, one of which is a molecule on the surface of an immune cell, such as an effector cell and/or an antigen presenting cell (APC).
In one embodiment, multispecific molecules of the invention include molecules comprised of at least one portion which binds to a molecule on an effector cell, such as an Fc receptor, and at least one portion (e.g., two, three, four or more portions) which binds to a different target, such as an antigen on a tumor cell or a pathogen. Therefore, these molecules can be used to induce effector cell-mediated elimination of a target.
In another embodiment, multispecific molecules of the invention include antigen xe2x80x9cmultimer complexesxe2x80x9d comprised of multiple (i.e., two or more) portions which bind to a molecule on an antigen presenting cell (APC), such as an Fc receptor, linked to one or more antigens. These multimer complexes target antigens, such as self-antigens, to APCs to induce and/or enhance internalization (endocytosis), processing and/or presentation of the antigen by the APC. Therefore, these molecules can be used to induce or enhance an immune response either in vivo or in vitro against a normally non-immunogenic protein, such as a self-antigen.
In another aspect, the invention features multispecific, multivalent molecules, which minimally comprise an anti-Fc receptor portion, an anti-target portion and optionally an anti-enhancement factor (anti-EF) portion. In preferred embodiments, the anti-Fc receptor portion is an antibody fragment (e.g., Fab or (Fabxe2x80x2)2 fragment), the anti-target portion is a ligand or antibody fragment and the anti-EF portion is an antibody directed against a surface protein involved in cytotoxic activity. In a particularly preferred embodiment, the recombinant anti-FcR antibodies, fragments or ligand are xe2x80x9chumanizedxe2x80x9d (e.g., have at least a portion of a complementarity determining region (CDR) derived from a non-human antibody (e.g., murine) with the remaining portion(s) being human in origin).
In another aspect, the invention features methods for generating multispecific molecules. In one embodiment, both specificities are encoded in the same vector and are expressed and assembled in a host cell. In another embodiment, each specificity is generated recombinantly and the resulting proteins or peptides are conjugated to one another via sulfhydryl bonding of the C-terminus hinge regions of the heavy chain. In a particularly preferred embodiment, the hinge region is modified to contain only one sulfhydryl residue, prior to conjugation.
In yet another aspect, the invention features molecular complexes, which comprise two or more binding specificities linked to at least one antigen. The binding specifities are for a component on the surface of an antigen presenting cell, which is capable mediating internalization of the molecular complex when bound by the binding specificities. In a preferred embodiment, the molecular complex comprises three or more binding specificities, and at least one of the binding specifities are specific to an Fc Receptor (e.g., Fcxcex3RI). In a particularly preferred embodiment, the antigen is non-immunogenic when administered in uncomplexed form.
The invention also features methods for inducing or enhancing an immune response against an antigen in a subject, by administering to the subject the molecular complex of the invention. Furthermore, the invention includes methods for immunizing a subject, by administering to the subject the molecular complex of the invention.
Recombinant antibodies and multispecific molecules generated therefrom can be engineered to have increased affinity and specificity. Further, humanized antibodies are typically less immunogenic when administered to a human. Other features and advantages of the present invention will become better understood by reference to the following Detailed Description and Claims.